Generally speaking, this invention relates to a system for establishing and maintaining an oxygen-free environment. More particularly, this invention relates to such a system which will establish such an oxygen-free environment in any sealed container, and which will regenerate such an environment subsequently on a repetitious basis when the container is unsealed for inspection and then resealed. As such, the system is a self-contained arrangement which has the ability not only to establish an anaerobic environment, but also to maintain and/or re-establish such an environment.
As will be appreciated, in the culturing of various media, it becomes important to inspect the state of the culture at various stages. In doing so, it becomes necessary to open the container in which the media is contained for such inspections, particularly when an anaerobic jar is utilized wherein several petri dishes are arranged or stacked. When the sealed container is opened, under those circumstances, it becomes necessary to re-establish the oxygen-free environment when the container is re-closed. This involves the utilization of additional catalysts and the insertion of an entirely new anaerobic generating system in order to re-establish the oxygen-free environment. Not only that, but all of the cultures are exposed and disturbed when the jar is opened, even though only one or two may need inspection at that time.
With this invention, by contrast, an arrangement is provided for re-establishing the oxygen-free environment over several repeated openings automatically with the resealing or reclosure of the container in which the culture media is contained. As such, separate small petri dishes, for example, may be utilized for the system of the invention here wherein each individual petri dish has self-contained therein a system for generating an oxygen-free environment which re-establishes itself automatically with reclosure of the container. Alternatively, when the couple is only moistened, the metal of the couple first liberates hydrogen which dries the environment and causes a continuous reaction with any free oxygen in the sealed environment. Thus, there is a continuous scavenging function when moisture is added in only a small quantity to the couple of the invention here. Thereafter, with the addition of water, the process is repeated with the generation of hydrogen at a moderate rate until all of the metal is used up.
Thus, this system, in accordance herewith, has a double action as an oxygen "scavenger." By placing approximately one and a half grams of the metallic couple inside a conventional petri dish or plate, for example, and exposing to water, either by high humidity as in pouring fresh molten culture media such as agar into the plate, or by direct addition of water to the couple, hydrogen is released to react with the free oxygen in the presence of a catalyst, also inserted into the container. This removes the free oxygen present in the environment. After this reaction has stopped, the dried metallic couple left will slowly react directly with any free oxygen left, and/or diffused out of the culture medium forming a metallic oxide, thus helping to maintain the environment in its reduced state. The formed metallic oxide will further dry the environment.
As will be appreciated by practitioners-in-the-art, each sealed environment, in accordance with the system herein, such as a petri dish or plate, will contain the metallic couple together with a catalyst such as palladium supported upon alumina, and an indicator for determining anaerobiosis. In addition, the container may include a carbon dioxide tablet for generating carbon dioxide. The indicator for indicating anaerobiosis may be methylene blue, indigo carmine or resazurin.
As discussed above, anaerobic petri plates or dishes may be opened, examined and then closed several times, and on each occasion, the plate will return to its reduced state with the addition of a small quantity of water. Thus, each plate automatically returns to its reduced state and maintains its environment during handling and observation.
Representative metals which may be utilized for carrying out this invention include magnesium, strontium and metallic calcium. Beryllium and barium may be used, but are extremely poisonous. Preferably, metallic magnesium is used in the form of magnesium turnings. The metallic salt added to the metal to form the metallic couple may be, for example, copper, platinum, palladium, tin, iron, nickel or cobalt. Preferably, it is a copper salt, and most preferably copper chloride. The catalyst for generating the anaerobiosis from this metallic couple may be, for example, a supported palladium or a supported platinum. However, it is within the purview of this invention that the metallic couple of the system herein may act as its own catalyst. If a metallic couple is formed from palladium and magnesium, for example, this combination may act as its own catalytic generating agent. Other salts which combine to act as their own catalytic generating agents include nickel, platinum and cobalt.
In forming the metallic couple of the system of the invention, a weak solution of cupric chloride may be provided which is poured through a funnel and over a portion of magnesium turnings. The coupling must be carried out rapidly in order to bring about completion of the reaction as soon as possible. In forming an anaerobic generating sealed container system, in accordance herewith, the quantity of copper-magnesium couple, for example, will be selected according to the volume of the container in which it is to be placed. For example, 0.5 grams of a copper-magnesium couple establishes a complete oxygen-free environment in a volume of 40 cc in about 2 hours. Anaerobic jars with a volume of 2.2 liters, for example, require 2 grams of a magnesium-copper couple in order to establish an anerobic atmosphere in a useful period of time for most culturing applications. The metallic couple system of the invention here is so effective, for example, that it will operate to establish anaerobiosis without a catalyst. The problem with operating under these conditions is that the reaction is too slow and will not be effective for certain very sensitive cultures.
Representative chemical reactions established for maintaining anaerobiosis in accordance herewith and assuming a copper-magnesium couple is being utilized, are as follows: ##STR1##
Thus, the invention here provides a self-contained petri dish or other sealed container containing an anaerobic environment and/or reduced media, with each petri dish being clearly visible and easily handled so that one or many such dishes may be run simultaneously without the need of a large bulky system such as anaerobic jars or anaerobic incubators, for example. Each petri dish may be handled separately without disturbing any other ones of the dishes being run while observing the one. The system of the invention provides a dual action system for removing oxygen in that first hydrogen is generated which catalytically reacts with oxygen present for establishing anaerobiosis, and secondly by combining directly with oxygen to form a metallic oxide. Each single anaerobic plate or dish, furthermore, has the ability to work several times, thus automatically generating hydrogen which is catalytically oxidized to water which allows the dish to be streaked, opened, closed, and opened repeatedly several times, with the re-establishment of the anaerobiosis. Moreover, because the couple in its moist or semi-dry state reacts slowly with oxygen which may be still present in the container, such a property serves to remove traces of residual oxygen from the medium and to maintain a reduced environment.
It will be appreciated, because of the relatively small space of individual sealed containers, and the capability of multiple openings, the exposure time to ambient is cut to a minimum with the re-estabishment of anaerobiosis within only several minutes. Because of this, clinical cultures can be taken, streaked immediately and taken to the laboratory so as to increase isolation efficiency and decrease the time lag between taking clinical specimens and the diagnosis and sensitivity results thereof.
As illustrative of procedures for formulating metallic couples for use in systems, in accordance with this invention, one may note the following examples. It is to be understood, however, that these examples are being presented with the understanding that they have no limiting character on their broad disclosure of the invention as generally set forth herein and as directed to men skilled in the art.